Understanding of West Nile virus (WNV) neutralization by antibodies comes from the study of WNV and its close relatives Saint Louis encephalitis virus, Murray Valley encephalitis virus (MVEV), and Japanese encephalitis virus, as well as more distant relatives such as dengue virus, yellow fever virus, and tick-borne encephalitis virus (TBEV). Strong similarities in the sequence of the flavivirus envelope proteins and the nearly identical position of the cysteines that form intra-molecular bonds within the envelope proteins (Nowak et al., 1987) suggest that the envelope proteins of all flaviviruses must have very similar structures. Therefore, information about any flavivirus is generally applicable to the others.
Heinz and Kunz (1982, 1977) showed that flaviviruses contain only three proteins, the envelope protein (E), the membrane protein (M), and the capsid protein (C). Recent structural studies of dengue virus (Kuhn et al., 2002) confirmed this and showed the physical relationship of these proteins in the virion. Only the E protein is exposed on the virion surface (Kuhn et al., 2002). Thus far, the three-dimensional structures of the E proteins for WNV, TBEV, and dengue have been solved (Kuhn et al., 2002; Mukhopadhyah et al., 2003; Rey et al., 1995). These have a finger-like structure with three clearly distinct domains, domain I being in the middle between domains II and III. Individual molecules of E protein lay flat across the virion surface with pairs of molecules lying beside each other in opposite orientation and three pairs laying side-by-side.
The flavivirus E protein is synthesized as part of a genome-length polyprotein that includes all viral proteins. It is subsequently released from the polyprotein by proteolytic cleavage. Early cleavages inside the polyprotein release the E protein still attached to the pre-membrane (pr) and M proteins, the combination of the pr and M proteins being known as the “prM” protein. The resulting prM-E protein is inserted into the endoplasmic reticulum membrane where it begins to fold into its mature conformation. The virus is assembled in intracellular compartments with the prM-E on the surface. Subsequent cleavages separate the E and prM proteins and cleave the prM to yield the mature M protein. The pr fragment is not incorporated into virions. The E protein may or may not have glycosylation sequences and therefore may or may not be glycosylated (Hanna, et al., 2005).
Flaviviruses infect cells by binding to the cell membrane, probably through an interaction between the RGD sequence of E protein domain III and cell-surface integrin (Lee et al., 2000), and entering through endosomes. When the endosome acidifies, the virion envelope proteins undergo extensive and irreversible changes in their intra- and inter-molecular conformation. The 180 individual E protein molecules disassociate from their dimers, reorient their domains and join to form 60 trimeric spikes that protrude from the virion membrane, insert the tip of the spikes into the endosomal membrane, and aggregate into 12 pentameric rings of trimeric spikes that fuse the virion membrane with the endosomal membrane, thus allowing the capsid to enter the cell's cytoplasm and begin replication (Bressanelli et al., 2004). It is clear that solubilization of the dimers from the virion surface ablates some neutralization-related epitopes (Heinz et al., 1991) but it is not clear how the rearrangement and trimerization alters E protein antigenic sites (Stiasny et al., 1996).
Since only the E protein is exposed on the virion surface, antibodies that bind to and neutralize intact, infectious virions must bind to the E protein. This has been proven by showing the development of neutralizing antibodies in animals immunized with proteins purified from virus (Heinz et al., 1990) and viral proteins produced in recombinant systems (Bray et al., 1989; Heinz et al., 1986; Heinz et al., 1982; Jan et al., 1993; Konishi et al., 1992; Mason et al., 1991; Men et al., 1991; Pincus et al., 1992; Schlesinger et al., 1992), and by passive protection experiments with monoclonal antibodies directed against the E protein (reviewed in Heinz et al., 1977, 1986; Roehrig 1986).
Antibodies that bind some areas on the E protein would be expected to neutralize the virus and antibodies that bind other areas might not. In order to discriminate between the neutralization activity of antibodies that bind the primary amino acid sequence from those that bind the secondary and tertiary structure of the properly folded E protein, Wengler and Wengler (1989) showed that reduction of disulfide bonds to destroy the protein's secondary and tertiary structure ablated the ability of WNV E protein to engender neutralizing antibodies. This experiment strongly suggested that neutralizing antibodies bind to the E protein secondary and tertiary conformational structure rather than linear structure. To confirm this, Roehrig et al. (1989) made peptides from MVEV E protein predicted epitopes and found that only one engendered neutralizing antibodies and only at a low level. Indeed, subsequent studies have shown that monoclonal antibodies usually bind either native E protein or denatured E protein and its peptides (Guirakhoo et al., 1989; Holzmann et al., 1993; Roehrig et al., 1989). Only antibodies that bind the native structure neutralize the virus.
To show exactly which areas of the E protein are attacked by neutralizing antibodies, mutations in viruses that have escaped neutralization by monoclonal antibodies were sequenced and mapped on the E protein surface (reviewed in Heinz et al., 1983; Heinz et al., 1990; Roehrig 1986). These data enabled the generation of crude structural models (Cammack et al., 1986; Kolaskar et al., 1999; Mandl et al., 1989; Roebrig et al., 1989; Roehrig et al.; 1983) that were subsequently refined to show that mutations mapped to all three structural domains defined by x-ray crystallographic methods (Cecilia et al., 1991; Gao et al., 1994; Hasegawa et al., 1992; Holzmann et al., 1997; Holzmann et al., 1993; Jiang et al., 1993; Lin et al., 1994; Mandl et al., 1989). This strongly suggests that antibodies can neutralize flaviviruses by binding to any of the three domains. Nevertheless, most studies have focused on domain III where many neutralizing monoclonal antibody escape mutations occur (Beasley et al., 2002). Domain III is also the binding site for some non-neutralizing antibodies (Sanchez et al., 2005). Domain III can be isolated from purified virions as a trypsin-resistant fragment (Winkler et al., 1987) or generated as a recombinant protein (Mason et al. 1989) but its reactivity with neutralizing monoclonal antibodies is dependent on the maintenance of its conformational structure by its single disulfide bond. Several antibodies appear to neutralize WNV by binding a peptide that is exposed on domain I only during the membrane fusion transition (Kanai et al., 2006) or a site that interferes with conformational changes in domain III (Nybakken et al., 2005).